The production of conifers from seed is greatly affected by Fusarium root rot, in which seedling roots are destroyed by infection with the soil-inhabiting fungus Fusarium. That disease, which can be caused by Fusarium oxysporum, F. oxysporum var. redolens, F. proliferatum, or F. solani, kills 20-90% of conifer seedlings grown in the Lake States region each year, and reduces the vigor and growth of infected seedlings which are not killed, as discussed in the 1983, 1985, and 1986 editions of Prey, et al., Forest Pest Conditions in Wisconsin, Annual report. Department of Natural resources, Div. of Resource Management, Bureau of Forestry, Madison, Wis. Fusarium can also cause damping-off disease, in which the stem of the seedling near the soil line is destroyed.
Current measures for controlling soil Fusarium spp. include soil fumigation with methyl bromide-chloropicrin in bareroot nurseries, and frequent fungicide applications to greenhouse-grown plants. These measures are often ineffective in controlling seedling Fusarium diseases because Fusarium spp. is often present in the seed. Also, methyl bromide-chloropicrin soil fumigation may not be allowed after the year 2000 because its use is viewed as an environmental hazard. R. S. Smith and S. W. Fraedrich (1993), "Back to the future--pest management without methyl bromide", Tree Planters' Notes 44:87-90. Thus, alternatives are needed for controlling Fusarium diseases in tree nurseries.
The use of biological control agents (living microorganisms used to control pests) is gaining recognition as an alternative disease control. The effective use of bacteria, actinomycetes, and fungi as agents for biological control of soil-borne plant disease has been demonstrated in several instances. Among the useful biological control bacteria are Bacillus megetarium, which controls Rhizoctonia solani on soybean, as disclosed in U.S. Pat. No. 5,403,583, and a mixture of combinations of three Pseudomonas spp. a Corynebacterium sp., and two Bacillus spp., which controls Aphanomyces root rot of peas, as disclosed in U.S. Pat. No. 5,244,658.
Actinomycetes are bacteria with fungus-like growth characteristics. Several isolates of the actinomycete Streptomyces have proved effective as biological control agents against soil-borne plant pathogens. A commercial product, Mycostop.RTM. biofungicide, contains an isolate of S. griseoviridis as its active ingredient. That product is effective as a seed and soil treatment against seed rots, root and stem rots, and wilt diseases of various ornamental plants, caused by Fusarium spp. and other fungi. M-L. Lahdenpera, et al. (1991), "Mycostop--A novel biofungicide based on Streptomyces bacteria", pp.258-263 in Biotic Interactions and Soil-Borne Diseases, A. B. R. Beemster, et al., eds., Elsevier, Amsterdam. The Mycostop.RTM. Biofungicide Directions for Use (Kemira Biotech, Helsinki, Finland) recommends Mycostop.RTM. for use on pine and other conifers.
Another Streptomyces sp. isolate, designated WYEC 108, disclosed in U.S. Pat. No. 5,403,584, is effective as a seed treatment against damping-off of chickpea caused by Pythium spp. That patent also described some inhibitory activity against Fusarium growing in agar-solidified growth media in petri plates.
Other Streptomycetes used for biological control include mutants of S. corchorusii and S. spirovirticillatus, effective against Fusarium wilt of French bean and the organism which causes bacterial wilt of banana (Pseudomonas solanacearum), described in El-Abyad, et al. (1993), "Inhibitory effects of UV mutants of Streptomyces corchorusii and Streptomyces spiroverticillatus on bean and banana wilt pathogens", Can. J. Bot. 71:1080-1086, and S. hygroscopicus var. geldanus, which controlled Rhizoctonia root rot of pea, caused by R. solani, described in C. S. Rothrock and D. Gottlieb (1984), "Role of antibiosis in antagonism of Streptomyces hygroscopicus var. geldanus to Rhizoctonia solani in soil", Can. J. Microbiol. 30:1440-1447.
Various fungi have been utilized as biological control agents to control fungal plant pathogens. Two yeasts, Pichia guilliermondii and Hanseniasporum uvarum, are effective in controlling preharvest and postharvest development of several pathogens on numerous commodities, as disclosed in U.S. Pat. No. 5,413,783. Several isolates of Trichoderma spp. have also been employed to control soil-borne diseases, as disclosed in U.S. Pat. Nos. 4,996,157 and 5,192,686, including Fusarium spp. on cotton, disclosed in U.S. Pat. No. 4,713,342.
Mycorrhizae are fungi which infect and form mutualistic relationships with plant roots. These fungi can improve plant growth by increasing the plant's assimilation of nutrients, especially phosphorus, which are sparingly soluble in the soil. Mycorrhizal infection will often make the plant roots more resistant to various soil-borne fungal pathogens. There are two major types of mycorrhizae: vesicular-arbuscular (VA) mycorrhizae, which infect most cultivated plants and produce specialized structures (vesicles or arbuscules) in the root cells, and ectomycorrhizae, which infect many forest trees such as pines and other conifers. Compositions and methods have been developed to help efforts to artificially inoculate plants with mycorrhizae. See, for example, U.S. Pat. Nos. 4,551,165 and 5,178,642. Also see a review of these efforts in M. A. Castellano (1994), "Current status of outplanting studies using ectomycorrhizae-inoculated forest trees", pp. 261-281 in Mycorrhizae and Plant Health, F. L. Pfleger and R. G. Linderman, eds., APS Press, St. Paul.
Ectomycorrhizal fungi are generally capable of infecting many species of plants. The ectomycorrhizal fungus which has been the most extensively investigated, Pitholithus sp., has been used to infect several species of the following woody plants: pine (Pinus), oak (Quercus), acacia (Acacia), and eucalyptus (Eucalyptus). Id. Additionally, many genera of ectomycorrhizal fungi, including Hebeloma and Laccaria, have been shown to be capable of infecting herbacious plants such as corn and wheat, as disclosed in U.S. Pat. No. 5,178,642. Thus, ectomycorrhizal fungi can be generally considered to be somewhat nonspecific in the plants they infect.
Both VA mycorrhizae and ectomycorrhizae have been utilized as biological control agents, with limited success. That work is reviewed in R. G. Linderman (1994), "Role of VAM fungi in biocontrol", pp. 1-25 Id., and L. C. Duchesne, "Role of ectomycorrhizal fungi in biocontrol", pp. 27-45 Id.
Ectomycorrhizae have shown some promise in controlling soil-borne diseases on conifer seedlings, but the protection to date has been unreliable due to the extreme variability of results. For example, Laccaria spp. exhibited limited control against Fusarium root rot and damping off on Douglas fir (described in N. E. Strobel and W. A. Sinclair (1991), "Influence of temperature and pathogen aggressiveness on biological control of fusarium root rot by Laccaria bicolor in douglas-fir", Phytopathol. 81:415-420) and pine (in P. Chakravarty and S. F. Hwang (1991), "Effect of an ectomycorrhizal fungus, Laccaria laccata, on Fusarium damping-off in Pinus banksiana seedlings", Eur. J. For. Path. 21:97-106, and Paxillus involutus increased resistance of pine seedlings by 47% to Fusarium root diseases, as described in L. C. Duchesne, et al. (1988), "Interaction between the ectomycorrhizal fungus Paxillus involutus and Pinus resinosa induces resistance to Fusarium oxysporum", Can. J. Bot. 66:558-562. Because of the limited and conditional control exhibited in these studies, the authors have expressed pessimism that they could be used effectively without further extensive research. See N. E. Strobel and W. A. Sinclair, supra, and L. C. Duchesne (1994), supra.
The present invention addresses a long felt need to provide an alternative to chemical control methods by utilizing a strategy employing novel ectomycorrhizae and Streptomyces isolates alone and in combination to effectively control conifer seedling diseases caused by Fusarium.